Objective:To investigate the effects of influenza A virus(IAV)on macrophages based on expression of cytokines mediated by Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathway,and to further explore the intervention effects of Ma Xing Shigan decoction drug containing serum.Methods:RAW264.7 was divided into control group,JAK/STAT signal pathway activator group,inhibitor group,model group,oseltamivir group,antiviral granule group and Ma Xing Shigan decoction group.Cell samples were collected after the intervention of IAV and subsequent treating of drug-containing serum for 24 and 48 hours.Secretion levels of IL-1β and CXCL2 in supernatant were detected by ELISA.mRNA expression levels of influenza virus Nuclear Pro-tein(NP),JAK1/2 and STAT1 of cells were detected by RT-qPCR.Protein expression levels of JAK1/2 and STAT1 were detected by Western blot,and protein expression levels of p-STAT1 was detected by immunofluorescence.Correlation between protein expression levels of p-STAT1 and secretion of IL-1β and CXCL2 were evaluated by Pearson correlation analysis.Results:Secretion levels of IL-1β and CXCL2,mRNA expression levels of NP,JAK1/2,STAT1,protein levels of JAK1/2,STAT1 and p-STAT1 were increased,and protein levels of p-STAT1 was positively correlated with the secretion of IL-1β and CXCL2.Ma Xing Shigan decoction could down-regulate secretions of IL-1β and CXCL2,mRNA expression levels of NP,JAK1/2 and STAT1,protein expression levels of JAK1,JAK2,STAT1 and p-STAT1.Conclusion:Activation of JAK1/2-STAT1 signal pathway and imbalance of inflammatory factors may be one of the molecular mechanisms of immunopathological damage of macrophages induced by IAV.Ma Xing Shigan decoction may alle-viate pathogenic effects of IAV by regulating this signal pathway.

AIM:This study aims to explore the mechanisms of influenza A virus(IAV)-induced macrophage inflammatory injury based on the interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3)signaling loop and investigate the intervention effects of Ma-Xing-Shi-Gan decoction(MXSGD)-medicated serum.METHODS:RAW264.7 and BV2 cells were divided into control,Janus kinase(JAK)/STAT signaling pathway activator,inhibitor,model,oseltamivir,antiviral particle,and MXSGD groups.After IAV modeling and serum interventions,the cells were cultured for 24 and 48 h,and the indicators were detected and analyzed.ELISA,RT-qPCR,Western blot,and immuno-fluorescence assay were used to detect the secretion levels of IL-6 in the cell culture supernatant,IL-6 and STAT3 mRNA expressions,protein expression of STAT3,and expression levels of phosphorylated STAT3(p-STAT3),respectively.Pearson correlation analysis was used to evaluate the correlation between p-STAT3 and IL-6 in the two cell types.A co-cul-ture model of the two cells was constructed,and the secretion levels of IL-6 in the cell culture supernatant was measured.Molecular docking analyses were performed for STAT3 and MXSGD.RESULTS:After IAV simulation,the secretion lev-els of IL-6 in the cell culture supernatant,mRNA expression levels of IL-6 and STAT3,and protein expression levels of STAT3 and p-STAT3 in both cell lines were elevated(P<0.05 or P<0.01).Pearson correlation analysis revealed that p-STAT3 expression was positively correlated with IL-6 expression.The secretion levels of IL-6 in the co-culture model in-creased(P<0.01).MXSGD down-regulated the secretion levels of IL-6 in the cell culture supernatant mRNA,expression levels of IL-6 and STAT3,and protein expression levels of STAT3 and p-STAT3 in two kinds of cells(P<0.05 or P<0.01),and inhibited the secretion levels of IL-6 in co-culture models.STAT3 demonstrated good binding energies for liquiritin,amygdalin,and ephedrine.CONCLUSION:IAV can induce inflammatory injury in macrophages,and its mechanism may be related to activation of the IL-6/STAT3 signaling loop.MXSGD may alleviate the pathogenic effects of IAV by modulating the signaling loop.

Objective:To investigate the effects of influenza A virus(IAV)on macrophages based on expression of cytokines mediated by Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathway,and to further explore the intervention effects of Ma Xing Shigan decoction drug containing serum.Methods:RAW264.7 was divided into control group,JAK/STAT signal pathway activator group,inhibitor group,model group,oseltamivir group,antiviral granule group and Ma Xing Shigan decoction group.Cell samples were collected after the intervention of IAV and subsequent treating of drug-containing serum for 24 and 48 hours.Secretion levels of IL-1β and CXCL2 in supernatant were detected by ELISA.mRNA expression levels of influenza virus Nuclear Pro-tein(NP),JAK1/2 and STAT1 of cells were detected by RT-qPCR.Protein expression levels of JAK1/2 and STAT1 were detected by Western blot,and protein expression levels of p-STAT1 was detected by immunofluorescence.Correlation between protein expression levels of p-STAT1 and secretion of IL-1β and CXCL2 were evaluated by Pearson correlation analysis.Results:Secretion levels of IL-1β and CXCL2,mRNA expression levels of NP,JAK1/2,STAT1,protein levels of JAK1/2,STAT1 and p-STAT1 were increased,and protein levels of p-STAT1 was positively correlated with the secretion of IL-1β and CXCL2.Ma Xing Shigan decoction could down-regulate secretions of IL-1β and CXCL2,mRNA expression levels of NP,JAK1/2 and STAT1,protein expression levels of JAK1,JAK2,STAT1 and p-STAT1.Conclusion:Activation of JAK1/2-STAT1 signal pathway and imbalance of inflammatory factors may be one of the molecular mechanisms of immunopathological damage of macrophages induced by IAV.Ma Xing Shigan decoction may alle-viate pathogenic effects of IAV by regulating this signal pathway.

AIM:This study aims to explore the mechanisms of influenza A virus(IAV)-induced macrophage inflammatory injury based on the interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3)signaling loop and investigate the intervention effects of Ma-Xing-Shi-Gan decoction(MXSGD)-medicated serum.METHODS:RAW264.7 and BV2 cells were divided into control,Janus kinase(JAK)/STAT signaling pathway activator,inhibitor,model,oseltamivir,antiviral particle,and MXSGD groups.After IAV modeling and serum interventions,the cells were cultured for 24 and 48 h,and the indicators were detected and analyzed.ELISA,RT-qPCR,Western blot,and immuno-fluorescence assay were used to detect the secretion levels of IL-6 in the cell culture supernatant,IL-6 and STAT3 mRNA expressions,protein expression of STAT3,and expression levels of phosphorylated STAT3(p-STAT3),respectively.Pearson correlation analysis was used to evaluate the correlation between p-STAT3 and IL-6 in the two cell types.A co-cul-ture model of the two cells was constructed,and the secretion levels of IL-6 in the cell culture supernatant was measured.Molecular docking analyses were performed for STAT3 and MXSGD.RESULTS:After IAV simulation,the secretion lev-els of IL-6 in the cell culture supernatant,mRNA expression levels of IL-6 and STAT3,and protein expression levels of STAT3 and p-STAT3 in both cell lines were elevated(P<0.05 or P<0.01).Pearson correlation analysis revealed that p-STAT3 expression was positively correlated with IL-6 expression.The secretion levels of IL-6 in the co-culture model in-creased(P<0.01).MXSGD down-regulated the secretion levels of IL-6 in the cell culture supernatant mRNA,expression levels of IL-6 and STAT3,and protein expression levels of STAT3 and p-STAT3 in two kinds of cells(P<0.05 or P<0.01),and inhibited the secretion levels of IL-6 in co-culture models.STAT3 demonstrated good binding energies for liquiritin,amygdalin,and ephedrine.CONCLUSION:IAV can induce inflammatory injury in macrophages,and its mechanism may be related to activation of the IL-6/STAT3 signaling loop.MXSGD may alleviate the pathogenic effects of IAV by modulating the signaling loop.

Objective:To invastigate the targets and signaling pathways of Gubiao Fanggan Modified Formula in regulating the defense against influenza A virus in immunosuppressed mice by network pharmacology,and the key targets were verified by immuno-suppressive mice model.Methods:TCMSP database was used to search the active ingredients of Gubiao Fanggan Modified Formula,and GeneCard,OMIM,PharmGkb databases were used to obtain the potential targets of the active ingredients to prevent influenza,and take their intersection targets were taken;STRING11.5 database was used to make protein-protein interaction network analyzed and finded the core targets;Cytoscape3.8.1 was used to build a traditional Chinese medicine-ingredient-disease target network,and GO enrichment analysis and KEGG enrichment analysis were performed.Intraperitoneal injection of cyclophosphamide was used to construct a mouse model of immune function suppression,normal group,model control group,Gubiao Fanggan Modified Formula group and oseltamivir group were set up,followed by prophylactic administration,and influenza virus intervention was performed on the fourth day.After 7 days of intragastric administration,the key targets were verified by mouse spleen index,HE staining,RT-qPCR and immunohistochemistry.Results:There were 82 active ingredients in five traditional Chinese medicines in Gubiao Fanggan Modi-fied Formula,and 72 common targets of drugs and diseases such as IL-6,TNF-α,IL-2,etc,mainly involving IL-17,TNF and AGE-RAGE signaling pathway.Gubiao Fanggan Modified Formula could increase spleen index and significantly reduce mRNA and protein expressions of IL-6 and TNF-α in spleen tissue of mice(P<0.05 or P<0.01).Conclusion:Gubiao Fanggan Modified Formula may regulate body's immune function through targets such as IL-6 and TNF-α,thereby preventing influenza virus infection.

AIM:To elucidate the possible biological mechanism of silica-induced acute lung injury in rats.METHODS:Sixteen Male Sprague-Dawley rats were divided into control and acute silicosis model groups,and instilled intratracheally with 1 mL of normal saline and 50 g/L silica suspension,respectively.After 7 d,the rats were sacrificed for collection of lung tissue and serum.The serum levels of interleukin-1β(IL-1β),IL-18 and tumor necrosis factor-α(TNF-α)were measured by using ELISA.The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and gasdermin D(GSDMD)were measured by immunohistochemistry.Bacterial DNA was ex-tracted from the lung tissue for 16S ribosomal RNA gene sequencing to characterize changes in the composition of lung flo-ra.The differences in the structure of bacterial flora between control and model groups were analyzed by bioinformatic analy-ses.RESULTS:Immunohistochemical analysis showed that the protein expression levels of NLRP3 and GSDMD were higher in the lungs of the rats in model group.In addition,serum cytokine profiling showed that IL-1β,IL-18 and TNF-α levels were significantly higher in model group.The most abundant bacterial genera in the lung flora of the rats in model group were Bifidobacterium,Clostridium sensu stricto 1,and Parasutterella.The NLRP3 and GSDMD levels in the lung tissue and IL-1β and TNF-α levels in serum were positively correlated with the abundance of Parasutterella.CONCLU-SION:The alterations in lung flora structure and increased inflammation levels may be the actual biological mechanisms underlying silica-induced acute lung injury.The modulation of lung flora may provide a basis for the prevention and treat-ment of silica-induced acute lung injury.

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide. Influenza A virus (IAV) has been found to activate multiple programmed cell death pathways, including ferroptosis. Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation, leading to cell death. However, little is known about how influenza viruses induce ferroptosis in the host cells. In this study, based on network pharmacology, we predicted the mechanism of action of Maxing Shigan decoction (MXSGD) in IAV-induced ferroptosis, and found that this process was related to biological processes, cellular components, molecular function and multiple signaling pathways, where the hypoxia inducible factor-1(HIF-1) signaling pathway plays a significant role. Subsequently, we constructed the mouse lung epithelial (MLE-12) cell model by IAV-infected in vitro cell experiments, and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage, increased reactive oxygen species (ROS) release, increased total iron and iron ion contents, decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4 (GPX4), increased expression of acyl-CoA synthetase long chain family member 4 (ACSL4), and enhanced activation of hypoxia inducible factor-1α (HIF-1α), induced nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in the HIF-1 signaling pathway. Treatment with MXSGD effectively reduced intracellular viral load, while reducing ROS, total iron and ferrous ion contents, repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway. Finally, based on animal experiments, it was found that MXSGD effectively alleviated pulmonary congestion, edema and inflammation in IAV-infected mice, and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.
Animals ; Mice ; Ferroptosis ; Network Pharmacology ; Reactive Oxygen Species ; Vascular Endothelial Growth Factor A ; Influenza A virus ; Iron ; Hypoxia

Objective:To investigate the relationship between the CT images of a lateral condylar tibial plateau fractures and complete medial collateral ligament (MCL) injury.Methods:Data of 59 patients with lateral condylar fracture of tibial plateau complicated with MCL injury admitted to Tianjin Hospital from January 2020 to November 2021 were collected, including 32 males and 27 females, aged 42.4±12.3 years (range, 19-65 years), there were 26 cases of extension injury and 33 cases of flexion injury. The patients were separated into two groups: those with partial MCL injury and those with total rupture based on preoperative MR examination and intraoperative valgus stress test following fracture fixation. According to the ABC fracture classification of lateral condyle tibial plateau proposed by Sun et al., the fracture locations were determined on CT images, and the lateral plateau collapse depth (LPD) was measured. The relationship between LPD and MCL complete rupture was analyzed by receiver operating characteristic (ROC) curve.Results:Among 59 patients with lateral condylar tibial plateau fracture and MCL injury, 42 had partial injuries and 17 had complete ruptures. According to the ABC fracture classification, there were 26 cases of extension injury (involving area A), 21 cases of AB type, and 5 cases of ABC type; and 33 cases of flexion type injury, 19 cases of B type, 12 cases of BC type, and 2 cases of C type. All the 17 cases of MCL complete fracture occurred in extension injury, including type AB (14 cases) and type ABC (3 cases). The difference between the mean LPDs of the MCL full rupture group and the partial injury group was not statistically significant ( t=0.11, P=0.567), and the mean LPDs of both groups were 11.7±5.3 mm (range, 4.3-28.1 mm) and 11.5±4.8 mm (range, 3.8-23.6 mm), respectively. The area under the curve (AUC) of the ROC curve analysis was 0.504, and there was no statistical correlation between lateral platform collapse depth and MCL injury. Among the 26 patients with extensional injury area, MCL was completely ruptures in 17 cases and partially injury in 9 cases, LPD was 11.7±5.3 mm (range, 4.3-28.1 mm) and 6.6±1.8 mm (range, 3.8-9.4 mm), respectively, and the difference was statistically significant ( t=3.57, P=0.009). The best predictive cut-off value of LPD was 7.25 mm, the sensitivity was 88.2%, the specificity was 77.8%, and the AUC was 0.868. Conclusion:When the lateral condyle fracture of the tibial plateau is located in the extensional injury area (involving the A area in the ABC fracture classification) and the LPD measured on the CT image is greater than 7.25 mm, the complete rupture of the MCL should be considered. Clinical MCL repair is required after the fracture fixation surgery for improved surgical outcomes.

Objective:To analyze the tendency of incidence and mortality of mechanical injuries among Chinese residents from 1990 through 2019 and to estimate the age-period-cohort effect.Methods:Based on the Global Burden of Disease (GBD) 2019 database, the incidence and mortality data of mechanical injuries among Chinese residents from 1990 to 2019 were extracted. The trends of age-standardized incidence rate (ASIR) and age-standardized death rate (ASDR) of mechanical injuries among Chinese residents by gender was analyzed using the joinpoint regression model, and the annual percent change (APC) and average annual percent change (AAPC) were calculated. The age-period-cohort model was used to quantitatively assess the effects of age, period, and cohort on ASIR and ASDR for mechanical injuries.Results:① Overall tendency: from 1990 to 2019, the ASIR of mechanical injuries showed an increasing trend (540.95/100 000 in 1990 vs. 815.34/100 000 in 2019), and the ASDR first increased slightly and then decreased (2.62/100 000 in 1990 vs. 2.87/100 000 in 2005 vs. 1.77/100 000 in 2019) among Chinese residents. During the observation period, ASIR and ASDR for mechanical injuries of male were higher than female. ② Joinpoint regression model analysis showed that the ASIR of mechanical injuries had a fluctuating trend of increasing first and then decreasing and then rising rapidly among Chinese residents from 1990 to 2019 (AAPC = 1.42%, t = 9.59, P < 0.001). The ASIR of the Chinese male showed a slight decrease and then continued to increase (AAPC = 1.47%, t = 8.72, P < 0.001), while the ASIR of the Chinese female showed a rapid rising at first, then rapidly declining and then rising again (AAPC = 1.31%, t = 12.11, P < 0.001). From 1990 to 2019, the ASDR of mechanical injuries showed a fluctuating downward trend of first decreasing, then increasing, and then rapidly decreasing among Chinese residents (AAPC = -1.39%, t = -6.72, P < 0.001). The decrease rate of ASDR among male was as same as that among all population (AAPC = -1.44%, t = -7.29, P < 0.001), but the decrease rate of ASDR in female was relatively slow (AAPC = -1.08%, t = -4.54, P < 0.001). ③ Age-period-cohort model analysis showed that, with the increase of age, the risk of mechanical injuries among the overall population, male and female in China increased, then decreased, and then increased rapidly. The first small peak was at the age of 45-49 years old in male and 65-69 years old in female. The overall death risk showed an increasing trend with age, with a slowly increasing trend before 75 years old, and a sudden increase after 75 years old. The peak age of death risk was between 90 and 94 years. In terms of period effect, the risk of mechanical injuries showed a gradually increasing trend with time among the overall population, male, and female in China, and the risk of death showed a trend of decreasing first and then rapidly increasing and then decreasing. In terms of cohort effect, the risk of mechanical injuries among the overall population, male, and female in China showed a gradual upward trend with the increase in the birth year, and the risk of death showed an M-shaped trend. Conclusions:From 1990 to 2019, the incidence of mechanical injuries showed an increasing trend, and the mortality increased first and then decreased. Although the disease burden has improved, it is still high. More attention needs to be paid to the prevention and control of mechanical injuries, especially in the young population.

Objective:To analyze the risk factors of deep venous thrombosis (DVT) in patients with tibial plateau fracture during preoperative period.Methods:From July 2017 to October 2019, a total of 264 patients undergoing surgeries of tibial plateau fractures were enrolled. Duplex ultrasonography (DUS) during hospitalization was used to screen for DVT of the bilateral lower extremities. Patients were divided into DVT group and non-DVT group according to results of DUS. Data on demographics, comorbidities, injury-related data, fracture type, laboratory biomarkers were collected and compared between groups with and without DVT.Results:The incidence of preoperative DVT was 39.0% (103/264) among 264 patients with traumatic tibial plateau fractures, and distal thrombosis predominated in DVT group. There were 103 cases in DVT group. 55 were males and 48 were females. The average age was 54.00±11.12. According to the Schatzker classification of tibial plateau fractures, 7 cases belonged to type I, 37 to type II, 2 to type III, 11 to type IV, 29 to type V, and 17 to type VI. There were 161 cases in non-DVT group. 89 were males and 72 were females. The average age was 48.57±13.25. According to the Schatzker classification of tibial plateau fractures, 23 cases belonged to type I, 76 to type II, 2 to type III, 10 to type IV, 33 to type V, and 17 to type VI. Univariate analysis showed that age ( t=3.451, P=0.001), the type of tibial plateau fracture ( χ2=8.314, P=0.004), D-dimer ( χ2=18.552, P<0.001), APTT ( t=2.869, P=0.004), ALB ( t=2.292, P=0.023) and Hb ( t=1.983, P=0.048) were statistically different than those in non-DVT group. Multivariate analysis showed age ( OR=1.033, 95% CI: 1.009, 1.058; P=0.007), the type of tibial plateau fracture ( OR=1.829, 95% CI: 1.014, 3.299; P=0.045) and D-dimer ( OR=1.914, 95% CI: 1.057, 3.464; P=0.032) were independent risk factors. Conclusion:The incidence of DVT in patients with tibial plateau fractures during preoperative period is high, and distal thrombosis is the main part of venous thrombosis of lower extremity. The type of tibial plateau fracture, age and the level of D-dimer are independent risk factors of preoperative DVT in patients with tibial plateau fractures.